The present invention relates to a process for preparing a compound of formula I, a GH secretagogue which can be used i.a. in treating medical disorders resulting from a deficiency in growth hormone.
Growth hormone is a hormone which stimulates growth of all tissues capable of growing. In addition, growth hormone is known to have a number of effects on metabolic processes, e.g., stimulation of protein synthesis and free fatty acid mobilisation and to cause a switch in energy metabolism from carbohydrate to fatty acid metabolism. Deficiency in growth hormone can result in a number of severe medical disorders, e.g., dwarfism.
Growth hormone is released from the pituitary. The release is under tight control of a number of hormones and neurotransmitters either directly or indirectly. Growth hormone release can be stimulated by growth hormone releasing hormone (GHRH) and inhibited by somatostatin. In both cases the hormones are released from the hypothalamus but their action is mediated primarily via specific receptors located in the pituitary. Other compounds which stimulate the release of growth hormone from the pituitary have also been described. For example arginine, L-3,4-dihydroxyphenylalanine (L-Dopa), glucagon, vasopressin, PACAP (pituitary adenylyl cyclase activating peptide), muscarinic receptor agonists and a synthetic hexapeptide, GHRP (growth hormone releasing peptide) release endogenous growth hormone either by a direct effect on the pituitary or by affecting the release of GHRH and/or somatostatin from the hypothalamus.
In disorders or conditions where increased levels of growth hormone is desired, the protein nature of growth hormone makes anything but parenteral administration non-viable. Furthermore, other directly acting natural secretagogues, e.g., GHRH and PACAP, are longer polypeptides for which reason parenteral administration is preferred. WO 97/23508 discloses a method for preparing the compound of formula I. This method is very expensive and makes use of reagents which are adverse to the environment.
It is an object of the present invention to provide a novel method for preparing a compound of formula I which method results in high yields and high purity. Moreover the present method is reproducible and more economic, and is suitable for large scale production.
In accordance with the present invention there is provided a novel and improved method for preparing a compound of formula I 
or a salt thereof, comprising
a) treating a compound of formula II 
xe2x80x83or a salt thereof, wherein P is a protecting group, with an agent capable of forming an amide or ester or mixed carbonic anhydride or anhydride or acid halide, in a solvent selected from an organic solvent or mixture of organic solvents or a mixture of organic solvent(s) and water, thereby producing and isolating a compound of formula III 
xe2x80x83or a salt thereof, wherein R together with the carbonyl moiety is an amide or ester or mixed carbonic anhydride or anhydride or acid halide,
b) treating the isolated compound of formula III, or a salt thereof, with a compound of formula (IV) 
xe2x80x83or a salt thereof, in a solvent selected from an organic solvent or mixture of organic solvents or a mixture of organic solvent(s) and water, thereby producing a compound of formula (V) 
xe2x80x83or a salt thereof, which is then deprotected in a conventional manner, to obtain the compound of formula (I) or a salt thereof.
Accordingly, the present invention relates to a method for preparing a compound of formula I 
or a salt thereof, comprising
a) treating a compound of formula II 
xe2x80x83or a salt thereof, wherein P is a protecting group, with an agent capable of forming an amide or ester or mixed carbonic anhydride or anhydride or acid halide, in a solvent selected from an organic solvent or mixture of organic solvents or a mixture of organic solvent(s) and water, thereby producing and isolating a compound of formula III 
xe2x80x83or a salt thereof, wherein R together with the carbonyl moiety is an amide or ester or mixed carbonic anhydride or anhydride or acid halide,
b) treating the isolated compound of formula III, or a salt thereof, with a compound of formula (IV) 
xe2x80x83or a salt thereof, in a solvent selected from an organic solvent or mixture of organic solvents or a mixture of organic solvent(s) and water, thereby producing a compound of formula (V) 
xe2x80x83or a salt thereof, which is then deprotected in a conventional manner, to obtain the compound of formula (I) or a salt thereof.
In one embodiment of the present method P is a group of formula xe2x80x94C(xe2x95x90O)xe2x80x94C1-12alkyl, xe2x80x94C(xe2x95x90O)xe2x80x94Oxe2x80x94C1-12alkyl, xe2x80x94C(xe2x95x90O)xe2x80x94C1-12alkenyl or xe2x80x94C(xe2x95x90O)xe2x80x94Oxe2x80x94C1-12alkenyl, optionally substituted with one or more halogen, C1-6alkyl, hetaryl, aryl or fused-ring aromatic system. P is preferably selected from xe2x80x94C(xe2x95x90O)H, Troc, Boc, and Fmoc. In one embodiment P is Troc. In a second embodiment P is Boc. In a third embodiment P is Fmoc. In a most preferred embodiment P is Troc.
In another embodiment of the present method the agent capable of forming an amide or ester or mixed carbonic anhydride or anhydride or acid halide is selected from benzotriazole, isobutyl chlorocarbonate, DHOBt, HOBt, HOSu, and HOAt. In a most preferred embodiment the agent capable of forming an amide or ester or mixed carbonic anhydride or anhydride or acid halide is DHOBt.
Furthermore the solvent in step b of the present method is preferably a mixture of an organic solvent and water, preferably an ester and water, such as C1-6alkyl-C(xe2x95x90O)xe2x80x94Oxe2x80x94C1-6alkyl, e.g. ethylacetate and water.
The de-protection is usually carried out by acidic, basic, oxidative or reductive cleavage, e.g. when the protection group is Troc then reductive cleavage is carried out with Zn and acetic acid.
In a preferred embodiment of the present method P is Troc and the agent capable of forming an amide or ester or mixed carbonic anhydride or anhydride or acid halide is DHOBt.
In another aspect the present invention relates to a compound of formula III 
or a salt thereof, wherein R together with the carbonyl moiety is an amide or ester or mixed carbonic anhydride or anhydride or acid halide, and P is a protecting group. In one embodiment P is a group of formula xe2x80x94C(xe2x95x90O)xe2x80x94C1-12alkyl, xe2x80x94C(xe2x95x90O)xe2x80x94Oxe2x80x94C1-12alkyl, xe2x80x94C(xe2x95x90O)xe2x80x94C1-12alkenyl or xe2x80x94C(xe2x95x90O)xe2x80x94Oxe2x80x94C1-12alkenyl, optionally substituted with one or more halogen, C1-6alkyl, hetaryl, aryl or fused-ring aromatic system. P is preferably selected from xe2x80x94C(xe2x95x90O)H, Troc, Boc, and Fmoc. In one embodiment P is Troc. In a second embodiment P is Boc. In a third embodiment P is Fmoc. In a most preferred embodiment P is Troc. In a further embodiment the agent capable of forming an amide or ester or mixed carbonic anhydride or anhydride or acid halide is selected from benzotriazole, isobutyl chlorocarbonate, DHOBt, HOBt, HOSu, and HOAt, preferably DHOBt. In a most preferred embodiment P is Troc and said amide or ester or mixed carbonic anhydride or anhydride or acid halide is obtained by reacting DHOBt with the carboxylic acid moiety in the compound of formula II.
In a further aspect the present invention relates to a compound of formula V 
or a salt thereof, wherein P is Troc or Fmoc.
In one embodiment the compound is 
In a second embodiment the compound is 
The compounds of the present invention, i.e. formula I, II, III, IV and V may optionally be on a salt form, such as a pharmaceutically acceptable salt form e.g. the pharmaceutically acceptable acid addition salts of compounds of formula I, II, III, IV and V, which include those prepared by reacting the compound of formula I, II, III, IV and V with an inorganic or organic acid such as hydrochloric, hydrobromic, sulfuric, acetic, phosphoric, lactic, malic, maleic, mandelic phthalic, citric, glutaric, gluconic, methanesulfonic, salicylic, succinic, tartaric, toluenesulfonic, trifluoracetic, sulfamic or fumaric acid and/or water. 
The process according to the present invention is illustrated in above scheme 1 and should in no way be interpreted as limiting the invention in any way. Thus, the above use of specific compounds are only for illustrative purposes.
The compound 1 is a commercially available compound or may be prepared from known starting materials by conventional reaction steps, for instance as illustrated in example 1, wherein 5-(N-Boc)-Amino-5-methyl-hex-(2E)-enoic acid disclosed in e.g. WO 97/23508 is converted to 5-(N-Troc)-Amino-5-methyl-hex-(2E)-enoic acid. It is possible to start from 5-(N-Boc)-Amino-5-methyl-hex-(2E)-enoic acid and introduce a suitable protecting group, such as a group of formula xe2x80x94C(xe2x95x90O)xe2x80x94C1-12alkyl, xe2x80x94C(xe2x95x90O)xe2x80x94Oxe2x80x94C1-12alkyl, xe2x80x94C(xe2x95x90O)xe2x80x94C1-12alkenyl or xe2x80x94C(xe2x95x90O)xe2x80x94Oxe2x80x94C1-12alkenyl, optionally substituted with one or more halogen, C1-6alkyl, hetaryl, aryl or fused-ring aromatic system, e.g. a protecting group selected from xe2x80x94C(xe2x95x90O)H, trichloroethyloxycarbonyl (Troc), tert-butyloxycarbonyl (Boc), and fluorenylmethyloxycarbonyl (Fmoc), preferably Troc, using a similar procedure as described in example 1 herein. Compound 1 is then reacted with an agent capable of forming an amide or ester or mixed carbonic anhydride or anhydride or acid halide, such as benzotriazole, isobutyl chlorocarbonate, 3,4-Dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine (DHOBt), N-hydroxy benzotriazol (HOBt), N-hydroxy succinimid (HOSu), and 1-hydroxy-7-azabenzotriazole (HOAt), preferably DHOBt, in a solvent selected from an organic solvent or mixture of organic solvents or a mixture of organic solvent(s) and water, such as ethylacetate, thereby producing the compound 2, which is isolated in a conventional manner, e.g. as described in connection with example 2 herein. The isolated compound 2, which is very crystallinic and thus easy to handle, is treated with compound 3, either as the free amine or as a salt, e.g. the HCl salt, in a solvent selected from an organic solvent or mixture of organic solvents or a mixture of organic solvent(s) and water, preferably a mixture of an organic solvent and water, such as an ether and water, e.g. THF and water, preferably an ester and water, such as C1-6alkyl-C(xe2x95x90O)xe2x80x94Oxe2x80x94C1-6alkyl, e.g. ethylacetate and water, thereby producing compound 4, which is then deprotected in a conventional manner, such as by reductive cleavage with an organic acid, such as acetic acid and Zn, or the like, to obtain the compound 5, which is the compound of formula 1. The compound 3 may be obtained as described in WO 97/23508.
In the above structural formulas and throughout the present specification, the following terms have the indicated meanings:
The C1-12-alkyl, C1-6-alkyl, or C1-4-alkyl groups specified herein are intended to include those alkyl or alkylene groups of the designated length in either a linear or branched or cyclic configuration. Examples of linear alkyl are methyl, ethyl, propyl, butyl, pentyl, hexyl and heptyl. Examples of branched alkyl are isopropyl, sec-butyl, tert-butyl, isopentyl, isohexyl and isoheptyl. Examples of cyclic alkyl are C3-12-cycloalkyl such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl. The C1-6-alkoxy groups specified herein are intended to include an oxygen atom connected to a C1-6-alkyl as defined above. Examples are methoxy, ethoxy, isopropyloxy, sec-butyloxy, tert-butyloxy, cyclopropyloxy, cyclobutyloxy, etc.
The term xe2x80x9carylxe2x80x9d is intended to include monovalent carbocyclic aromatic ring moieties, being either monocyclic, bicyclic or polycyclic, e.g. selected from the group consisting of phenyl and naphthyl, optionally substituted with one or more C1-6-alkyl, C1-6-alkoxy, halogen, amino or aryl. Such aryl groups are described in Morrison and Boyd xe2x80x9cOrganic Chemistryxe2x80x9d, 4. Ed.
The term xe2x80x9chetarylxe2x80x9d is intended to include monovalent heterocyclic aromatic ring moieties, being either monocyclic, bicyclic or polycyclic, e.g. selected from the group consisting of pyridyl, 1-H-tetrazol-5-yl, thiazolyl, imidazolyl, indolyl, pyrimidinyl, thiadiazolyl, pyrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thienyl, quinolinyl, pyrazinyl, or isothiazolyl, optionally substituted by one or more C1-6alkyl, C1-6-alkoxy, halogen, amino or aryl. Such hetaryl groups are described in Morrison and Boyd xe2x80x9cOrganic Chemistryxe2x80x9d, 4. Ed.
The term xe2x80x9cfused-ring aromatic systemxe2x80x9d is intended to include monovalent aromatic ring moieties, being either bicyclic or polycyclic hydrocarbons, such as a system selected from the group consisting of fluorenes, e.g. flourenyl. Such fused-ring aromatic systems are described in Morrison and Boyd xe2x80x9cOrganic Chemistryxe2x80x9d, 4. Ed.
The term xe2x80x9chalogenxe2x80x9d is intended to include chlorine (Cl), fluorine (F), bromine (Br) and iodine (I).
The term xe2x80x9cProtecting groupxe2x80x9d is intended to include any group which protect the amino group when the carboxylic group in the compound of formula II is subjected to functional derivatization, and which is easy to remove afterwards by cleavage. Such protecting groups are described in xe2x80x9cProtective groups in organic chemistryxe2x80x9d, 2. Ed, Greene, T. W.; Wuts, P. G. M., John WileyandSons,Inc. 1991; and xe2x80x9cThe Peptides, Analysis, Synthesis, Biologyxe2x80x9d, vol 3 xe2x80x9cProtection of Functional Groups in Peptide synthesisxe2x80x9d, Gross, E.; Meienhofer, J.; Academic Press. A suitable protecting group is a group of formula xe2x80x94C(xe2x95x90O)xe2x80x94C1-12alkyl, xe2x80x94C(xe2x95x90O)xe2x80x94Oxe2x80x94C1-12alkyl, xe2x80x94C(xe2x95x90O)xe2x80x94C1-12alkenyl or xe2x80x94C(xe2x95x90O)xe2x80x94Oxe2x80x94C1-12alkenyl, optionally substituted with one or more halogen, C1-6-alkyl, hetaryl, aryl or fused-ring aromatic system, e.g. Troc, Boc, and Fmoc.
The term xe2x80x9cagent capable of forming an amide or ester or mixed carbonic anhydride or anhydride or acid halidexe2x80x9d is intended to include such agents which activates the compound by forming a functional derivative which may be used in acylation. Such agents are described in xe2x80x9cThe Peptides, Analysis, Synthesis, Biologyxe2x80x9d, vol 1 xe2x80x9cMajor Methods of Bond Formationxe2x80x9d, Gross, E.; Meienhofer, J.; Academic Press, 1981. Suitable agents are selected from benzotriazole, isobutyl chlorocarbonate, DHOBt, HOBt, HOSu, and HOAt.
The term xe2x80x9cde-protectionxe2x80x9d is intended to include acidic, basic, oxidative or reductive cleavage as described in xe2x80x9cThe Peptides, Analysis, Synthesis, Biologyxe2x80x9d, vol 1 xe2x80x9cMajor Methods of Bond Formationxe2x80x9d, Gross, E.; Meienhofer, J.; Academic Press, 1981. When, for instance, the protection group is Troc then reductive cleavage is carried out with Zn and acetic acid.
The compounds of formula I when used for the intended purpose of releasing endogenous growth hormone may be administered in pharmaceutically acceptable acid addition salt form or, where appropriate, as a alkali metal or alkaline earth metal or lower alkylammonium salt. Such salt forms are believed to exhibit approximately the same order of activity as the free base forms.
The compounds of the general formula I possess the ability to release endogenous growth hormone in vivo as mentioned in WO 97/23508. The compounds may therefore be used in the treatment of conditions which require increased plasma growth hormone levels such as in growth hormone deficient humans or in elderly patients or livestock.
To those skilled in the art, it is well known that the current and potential uses of growth hormone in humans are varied and multitudinous. Thus, compounds of formula I can be administered for purposes stimulating release of growth hormone from the pituitary and would then have similar effects or uses as growth hormone itself. Compounds of formula I are useful for stimulation of growth hormone release in the elderly, prevention of catabolic side effects of glucocorticoids, prevention and treatment of osteoporosis, treatment of chronic fatigue syndrom (CFS), treatment of acute fatigue syndrom and muscle loss following election surgery, stimulation of the immune system, acceleration of wound healing, accelerating bone fracture repair, accelerating complicated fractures, e.g. disctraction osteogenesis, treatment of wasting secondary to fractures, treatment of growth retardation, treating growth retardation resulting from renal failure or insufficiency, treatment of cardiomyopathy, treatment of wasting in connection with chronic liver disease, treatment of thrombocytopenia, treatment of growth retardation in connection with Crohn""s disease, treatment of short bowel syndrome, treatment of wasting in connection with chronic obstructive pulmonary disease (COPD), treatment of complications associated with transplantation, treatment of physiological short stature including growth hormone deficient children and short stature associated with chronic illness, treatment of obesity and growth retardation associated with obesity, treatment of anorexia, treatment of growth retardation associated with the Prader-Willi syndrome and Tumer""s syndrome; increasing the growth rate of a patient having partial growth hormone insensitive syndrome, accelerating the recovery and reducing hospitalization of burn patients; treatment of intrauterine growth retardation, skeletal dysplasia, hypercortisolism and Cushing""s syndrome; induction of pulsatile growth hormone release; replacement of growth hormone in stressed patients, treatment of osteochondrodysplasias, Noonan""s syndrome, schizophrenia, depressions, Alzheimer""s disease, delayed wound healing and psychosocial deprivation, treatment of catabolism in connection with pulmonary dysfunction and ventilator dependency, treatment of cardiac failure or related vascular dysfunction, treatment of impaired cardiac function, treatment or prevention of myocardial infarction, lowering blood pressure, protection against ventricular dysfunction or prevention of reperfusion events, treatment of adults in chronic dialysis, attenuation of protein catabolic responses after major surgery, reducing cachexia and protein loss due to chronic illness such as cancer or AIDS; treatment of hyperinsulinemia including nesidioblastosis, adjuvant treatment for ovulation induction; stimulation of thymic development and prevention of the age-related decline of thymic function, treatment of immunosuppressed patients, treatment or sarcopenia, treatment of wasting in connection with AIDS, improvement in muscle strength, mobility, maintenance of skin thickness, treatment of metabolic homeostasis and renal homeostasis in the frail elderly, stimulation of osteoblasts, bone remodelling and cartilage growth, regulation of food intake, stimulation of the immune system in companion animals and treatment of disorders of aging in companion animals, promoting growth in livestock and stimulation of wool growth in sheep, increasing milk production in livestock, treatment of metabolic syndrome (syndrome X), treatment of insulin resistance, including NIDDM, in mammals, e.g. humans, treatment of insulin resistance in the heart, improvement of sleep quality and correction of the relative hyposomatotropism of senescence due to high increase in REM sleep and a decrease in REM latency, treatment of hypothermia, treatment of frailty associated with aging, treatment of congestive heart failure, treatment of hip fractures, treatment of immune deficiency in individuals with a depressed T4/T8 cell ratio, treatment of muscular atrophy, treatment of musculoskeletal impairment in elderly, enhancing the activity of protein kinase B (PKB), improvement of the overall pulmonary function, and treatment of sleep disorders. Treatment is also intended to include prophylactic treatment.
For the above indications the dosage will vary depending on the compound of formula I employed, on the mode of administration and on the therapy desired. The dosage of the compounds according to this invention is suitably 0.01-500 mg/day, e.g. from about 5 to about 50 mg, such as about 10 mg per dose, when administered to patients, e.g. humans, as a drug. However, generally dosage levels between 0.0001 and 100 mg/kg body weight daily are administered to patients and animals to obtain effective release of endogenous growth hormone. Morever the compounds of formula I have no or substantially no side-effects, when administered in the above dosage levels, such side-effects being e.g. release of LH, FSH, TSH, ACTH, vasopressin, oxytocin, cortisol and/or prolactin. Usually, dosage forms suitable for oral, nasal, pulmonal or transdermal administration comprise from about 0.0001 mg to about 100 mg, preferably from about 0.001 mg to about 50 mg of the compounds of formula I admixed with a pharmaceutically acceptable carrier or diluent.
Optionally, a pharmaceutical composition comprising the compound of formula I prepared by the method of the invention may be combined with one or more compounds exhibiting a different activity, e.g., an antibiotic or other pharmacologically active material.
The route of administration may be any route which effectively transports the active compound to the appropriate or desired site of action, such as oral, nasal, pulmonary, transdermal or parenteral, the oral route being preferred.
Apart from the pharmaceutical use of the compounds of formula I, they may be useful in vitro tools for investigating the regulation of growth hormone release.
Compounds of formula I may also be useful in vivo tools for evaluating the growth hormone releasing capability of the pituitary. For example, serum samples taken before and after administration of these compounds to humans can be assayed for growth hormone. Comparison of the growth hormone in each serum sample would directly determine the ability of the patients pituitary to release growth hormone.
Compounds of formula I may be administered to commercially important animals to increase their rate and extent of growth, and to increase milk production.
A further use of growth hormone secretagogue compounds of formula I is in combination with other secretagogues such as GHRP (2 or 6), GHRH and its analogues, growth hormone and its analogues or somatomedins including IGF-1 and IGF-2.